The theory of Fitch and Markowitz for concomitantly variable codons (covarions) in evolution predicted existence of functional correlation in amino acid residue replacements between present day cytochromes c (cyt. c). Mutational analysis has been carried out on yeast iso-2 cyt. c since previous years, where hydrophobic core residues I20, M64, L85, M98 and surface residue L9 were replaced in various combinations with those found in mammalian and bird cyt. c. The function assay is based on the ability of yeast cells to grow in YPGE medium. Furthermore, experiments, in previous years, on the single M64L and M98L mutations as well as the double M64L/M98L using NMR showed that the effects of these mutations is to perturb the structural integrity of the protein. We identified functional correlation in two cases of a pair of residue replacements, the I20 to V and M98 to L pair, and the L9 to I and L85 to I. In both cases, only one of the two alternative, putative evolutionary pathways leads to a functional protein and the corresponding pairs of residue replacements are among those residues found in known cyt. c. Since valine is predicted to be at position 20 in the ancestral form of cyt. c, these data provide an explanation for the ancient requirement of leucine rather than methionine in position 98. These data provide further evidence for the role of those specific atom-atom interactions in directing a pathway in evolutionary changes of the amino acid sequence that have taken place in cyt. c, in accordance with the covarion theory. Anti-yeast iso-1 cyt.c monoclonal antibodies 2-96-12 and 4-74-6 have closely related epitopes. However, while the specificity of 4-74-6 is stringent, 2-96-12 cross-reacts with many evolutionarily related cyt. c. Such a marked diference in specificity of antibodies with overlapping epitopes may represent unique antibody immuno diversity. Thus, we constructed, in previous years, Fv fragment models consisting of the variable domains of the heavy and light chains of 2-96-12 and 4-74-6 and that of another anti- iso-1 cyt. c as a control to gain insight into the origin of this difference in specificity. Our models show that 4-74-6 and 2-96-12 contain five and two aromatic side chains, respectively in or near the central area of the antigen combining site. The side chains of Arg95H (heavy chain) in 2-96-12 and Arg91L (light chain) in 4-74-6 project toward the central area of the combining site in our model. Antigen docking to our Fv models, combined with previous immunological studies, suggests that both epitopes of 2-96-12 and 4-74-7 may include iso-1 cyt. c Leu58, Asp60, Asn62, and Asn63 and that Asp60 may interact with Arg95H in 2-96-12 and Arg91L in 4-74-6. The effect of the Arg95H to Lys mutation on the antigen binding found previously is also in accord with our model. The difference in specificity may be partly explained by a greater degree of conformational flexibility in and around the central area of the combining site in 2-96-12 compared to 4-74-6 due to differences in aromatic side chain packing. - Evolution, Cytochrome c, Mutagenesis, Covarion theory, Antibody immuno diversity, Combining site model